1. Field of the Invention
The present invention relates to a cell-free matrix-gel graft for tissue regeneration and, in particular, for cartilage regeneration, a method for its production and the use of the graft for tissue regeneration.
2. Discussion of the Background
The articular cartilage is indispensable as sliding surface for normal joint function. Damage to the articular cartilage occurs for example in osteoarthrosis, trauma or osteochondritis dissecans. The articular cartilage cells of which the articular cartilage is composed have only low regeneration ability in adults.
The articular cartilage is a mesodermal tissue type which is derived from connective tissue and which can be ascribed to multipotent, undifferentiated mesenchymal progenitor cells. Hyaline cartilage is the most widespread type of cartilage and is found for example in the joint surfaces. Cartilage defects owing to wear or damage represent a widespread medical problem. For this reason, in the past, especially in recent years, methods and techniques have been developed for replacing defective chondral or else osteochondral areas in the articular cartilage. Thus, periosteal, perichondral, allogeneic and autologous osteochondral grafts, allogeneic menisci or else prostheses made of synthetic materials have been employed as replacement for articular cartilage.
In the autologous grafting of chondrocytes, chondrocytes taken from the patient are expanded in cell culture and returned to the patient. A wide variety of types of graft is possible for the return. Examples thereof are injection solutions injected into the joint, matrices inoculated with cartilage cells, and the like.
For example, WO 97/15655 describes artificial tissues consisting of three-dimensional extracellular matrices and genetically manipulated cells, where the matrices are able to release immunosuppressive or cell-differentiating factors. The matrices are preferably in the form of a polymer web into which a cell suspension, which may for example be suspended in a fibrinogen solution, is dispersed. It is additionally possible to add to the matrix factors or components of the appropriate extracellular matrix which promote the growth and/or differentiation process. In order to keep the cells in the matrix, the cell suspension can be consolidated by adding thrombin in order to obtain the finished graft.
DE 44 31 598 describes a method for producing an implant from cell cultures, in which three-dimensional support structures on which cells are deposited are initially enveloped and then perfused with a nutrient solution. Absorbable microparticles are incorporated into the support structures and release factors which influence tissue formation during absorption.
DE 100 06 822 describes a method for producing a bone or cartilage graft in which bioabsorbable and biocompatible framework structures consist of osteogenic cells crosslinked by fibrin or hydrogel, and factors, and have been shaped to geometric articles which can be fitted together.
DE 43 06 661 describes a three-dimensional support structure, preferably made of a polymer web, into which cells are incorporated. The support structure is then perfused in nutrient solution in order to promote cell growth and the formation of an extracellular matrix by the cells. The support structure is enveloped with agarose in order to prevent the cells migrating out or being washed out.
DE 101 39 783 further discloses the provision of mesenchymal cells in synovial fluid. This composition can, if desired, also be applied to a support such as a web or a synthetic material and be used in this form as graft. Otherwise, the suspension of cells in synovial fluid is injected as such into the affected joint.
Alternatively, matrix structures which themselves do not contain any cells are synthesized. Thus, for example, US 2003/0003153 describes reinforced matrix membranes which comprise one or more scaffold-forming proteins which are suitable for cell growth. It is assumed in these cases that cells from endogenous tissue will migrate into the matrix structure. This is achieved for example by conventional Pridie perforation or microfracturing. In these techniques, slight perforations or fractures are introduced into the bone of the joint as far as the bone marrow. Bleeding occurs through the perforations into the defect, thus filling the defect with a blood clot. Mesenchymal progenitor cells are present in the clot and, when stimulated by appropriate stimuli, are able to form a cartilage-like replacement tissue, called fibrous cartilage. If a matrix material is provided over the Pridie perforation, the blood cells are able to migrate into this matrix material and settle there.
DE 199 57 388 and WO 2005/014027 make use of this effect and enhance it by providing growth and differentiation factors (DE 199 57 388), chemokines (WO 2005/014027) or blood serum (DE 10 2005 030 614) as recruiting agents in the matrix structure. All the factors are intended to lead to enhanced recruitment of the cartilage-forming mesenchymal progenitor cells, the ultimate intention being to achieve faster regeneration of the cartilage.
WO 02/00272, finally, discloses the possibility of producing corresponding grafts also from blood and a polymer component. This document addresses the problem that the blood clot normally produced in the Pridie perforation contracts on coagulation and thus changes shape. The added polymer prevents this change in shape and thus permits true-shape healing. The graft is produced by a polymer being mixed with blood or a blood component such as erythrocytes, leukocytes, monocytes, platelets, fibrinogen, thrombin and platelet-rich plasma and being introduced into the defect. However, when a blood component is used it is essential that material capable of coagulation is present in order to achieve the desired effect.
US2005/0043814 finally discloses a cell-free matrix implant with optional bone-inducing composition, which includes a collagenous, thermo-reversible gel, an aromatic organic acid or an adsorbable caprolactone polymer support matrix. The bone-inducing composition may comprise polyglycolic acid polymers and be applied to a matrix of collagens or polyglycolic acids. When this cell-free matrix implant is used, it is essential that the gel is thermo-reversible because this makes it possible for the composition to be applied in liquid form. Only after injection of the liquid composition does it solidify in the patient's body.
The disadvantages of the technologies described above are that if the graft itself comprises cells, these are frequently damaged by the manipulation during handling, the graft has to be produced by a lengthy culturing method on use of cells, especially autologous cells, and requires careful checks on contamination, and finally storability is lacking, or storage is possible only under complicated conditions.
These disadvantages are further enhanced on use of allogeneic, exogenous cells to the extent that an elaborate bacteriological and virological investigation of the donor cells is necessary in order to avoid transmission of diseases by the cell-containing graft. In addition there is the danger of a rejection response on use of exogenous cells.
Further disadvantages of the technologies described above are that factors which accelerate or enable the migration in of cells are added to cell-free grafts. These factors may be for example either growth and differentiation factors or chemokines. These factors are of animal origin, i.e. isolated from animals, or are produced recombinantly by bacteria or yeasts. However, the factors produced on recombinant production are predominantly derived from a structure of animal origin. This is disadvantageous because, owing to the difference between “donor” and “recipient” of these constituents of the graft, it is easily possible for incompatibilities or allergic reactions to occur after the grafting.
The use of blood, blood components or serum for recruiting cells into the cell-free graft is inadequate in as much as elaborate bacteriological and virological investigations are likewise necessary on use of allogeneic, exogenous blood to avoid transmission of diseases. On the other hand, a precondition for the use of endogenous blood, blood components or serum is additional manipulation on the graft (introduction of the component) and on the patient (taking of blood). Every manipulation on the graft entails the risk of contamination of the graft, which may likewise lead to incompatibility in the patent. In addition, the additional removal of material from the patient necessary for this is associated with undesirably long times required and additional costs.
The disadvantage of the use of implants which solidify after implantation is that a solid, set implant mechanically damages the surrounding tissue of lower strength/hardness. In addition, a solid implant impedes the migration in of cells and requires very long break-down times and absorption times.
A further disadvantage of the cell-free grafts described above is that in the background art they are employed after a Pridie perforation or microfracturing in order to accommodate nonselectively all the cells which have been introduced or imported during the bleeding into the cell-free graft. This may result in overgrowth of the graft with cells and/or constituents not typical of the tissue, which might impede the formation of the desired tissue or promote the formation of a mixed tissue. Such a bleeding into the defect is disadvantageous in as much as it may lead to irritation and inflammation of the surrounding tissue. Thus, Hooiveld and colleagues describe damage to cartilage cells resulting from bringing cartilage together with blood or blood components for 4 days [Hooiveld M. J. et al.: Haemoglobin-derived iron-dependent hydroxyl radical formation in blood-induced joint damage: an in vitro study, Rheumatology 43, 784-790, 2003]. Hooiveld further describes the possibility of internal synovial membrane inflammation (synovitis) being caused by bleeding into the joint [Hooiveld M. J. et al.: Immature articular cartilage is more susceptible to blood-induced damage than mature articular cartilage: an in vivo animal study, Arthritis Rheum 48, 396-403, 2003].
It is particularly disadvantageous with the grafts known in the background art that, because of the cells or biological constituents present, they can be stored for only a very limited time and additionally require very specific storage conditions.
The present invention has the object inter alia of providing a graft which is simple to produce, requires the minimum number of manipulation steps for production and can be stored very easily. It was additionally intended that it can be used rapidly and simply, but nevertheless ensure comparable and/or at least as good therapeutic results as the grafts known in the background art. It would further be desirable to be able to dispense as far as possible with the use of exogenous, where appropriate even recombinant growth factors, which potentially represent allergens. It would also be desirable to be able as far as possible to dispense with additional removal of blood or the use of blood, blood components or serum, because this represents an additional risk of contamination and stress for the patient. It would further be desirable to be able to prevent as far as possible the bleeding into the defect after Pridie perforation or microfracturing, in order to be able to avoid damage to the surrounding articular tissue. It would also be desirable for the cell-free graft to have the strength or elasticity of the surrounding tissue, in order to prevent mechanical damage to the surrounding tissue. In this context a possible adaptation of the hardness/elasticity of the graft to the individual patent in order to minimize unharmonic movements caused by different hardnesses of the materials would be desirable.